Bottom Line:
Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents.Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence.Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential.

ABSTRACTSpectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

fig2: Distribution of polarization-resolved fluorescence intensities and corresponding anisotropy values of DASPMI-stained XTH2 cells. The very distinct high anisotropy at high-intensity regions (inserts) inside several individual mitochondria is apparent. Such submitochondrial zones of higher membrane potential are known to exist from previous studies in XTH2 cells. The plot of mean anisotropy values and corresponding pixel fluorescence intensities (± 50 units) were fitted to an exponential function of the form

Mentions:
Representative images of the distribution of polarization-resolved fluorescence of DASPMI in mitochondria of living XTH2 cells are shown in Fig. 2. Apart from regions of higher intensity near the nucleus, submitochondrial zones of higher membrane potential revealed from higher DASPMI intensity were found to have higher anisotropy as well (arrowheads in inserts in Fig. 2). The presence of submitochondrial zones with higher membrane potential has been observed previously using DASPMI and JC1 dyes (17,18).

fig2: Distribution of polarization-resolved fluorescence intensities and corresponding anisotropy values of DASPMI-stained XTH2 cells. The very distinct high anisotropy at high-intensity regions (inserts) inside several individual mitochondria is apparent. Such submitochondrial zones of higher membrane potential are known to exist from previous studies in XTH2 cells. The plot of mean anisotropy values and corresponding pixel fluorescence intensities (± 50 units) were fitted to an exponential function of the form

Mentions:
Representative images of the distribution of polarization-resolved fluorescence of DASPMI in mitochondria of living XTH2 cells are shown in Fig. 2. Apart from regions of higher intensity near the nucleus, submitochondrial zones of higher membrane potential revealed from higher DASPMI intensity were found to have higher anisotropy as well (arrowheads in inserts in Fig. 2). The presence of submitochondrial zones with higher membrane potential has been observed previously using DASPMI and JC1 dyes (17,18).

Bottom Line:
Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents.Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence.Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential.

ABSTRACTSpectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.